Validated method for the determination of idazoxan in human plasma by liquid chromatography with tandem mass spectrometric detection

The effect of perchlorate anion as a mobile phase modifier on the retention of dansyl norvaline and dansyl tryptophan enantiomers on a human serum albumin (HSA) column was studied by varying the chaotropic agent concentrations. The thermodynamic parameters for the transfer of a solute from the mobile to the HSA stationary phases were determined from linear van't Hoff plots. An enthalpy-entropy compensation study revealed that the type of interaction between the solute and HSA was independent of the molecular structure. The parabolic variations observed with the enthalpic and entropic terms of dansyl amino acid transfer in relation to the concentration of perchlorate anion were considered to be the result of the change from reversed to normal-phase conditions for this chromatographic system.     

Automated determination of tramadol enantiomers in human plasma using solid-phase extraction in combination with chiral liquid chromatography

Age-related changes in the facial expression of pain during the first 18 months of life have important implications for our understanding of pain and pain assessment. We examined facial reactions video recorded during routine immunization injections in 75 infants stratified into 2-, 4-, 6-, 12-, and 18-month age groups. Two facial coding systems differing in the amount of detail extracted were applied to the records. In addition, parents completed a brief questionnaire that assessed child temperament and provided background information. Parents' efforts to soothe the children also were described. While there were consistencies in facial displays over the age groups, there also were differences on both measures of facial activity, indicating systematic variation in the nature and severity of distress. The least pain was expressed by the 4-month age group. Temperament was not related to the degree of pain expressed. Systematic variations in parental soothing behaviour indicated accommodation to the age of the child. Reasons for the differing patterns of facial activity are examined, with attention paid to the development of inhibitory mechanisms and the role of negative emotions such as anger and anxiety.     

Analysis of human milk triacylglycerols by high-performance liquid chromatography with light-scattering detection

A high-performance liquid chromatography (HPLC) method for the separation of human milk triacylglycerols using a C18 Spherisorb ODS column and ternary gradient elution with dichloromethane, acetone and acetonitrile is described. The triacylglycerols are detected by light scattering. Several chromatographic conditions were assayed in order to optimize the method: sample solubility, mobile phase, column temperature and the mass detector oven temperature. The linearity, precision and relative response of the method were examined. A total of 34 peaks were separated and quantified based on the percentage peak area in the HPLC chromatogram. Mature human milk analyzed by this method contained six predominant triacylglyceride structures: POO, POL, LOO, POP, OOO and SOP, where P = palmitin, O = olein, L = linolein and S = stearin.     

Purification of human immunoglobulin M by affinity chromatography on protamine-Sepharose

Human IgM has been isolated from plasma by a simple procedure in high yield. The first step was adsorption to protamine-Sepharose and elution by increasing the ionic strength with NaCl. This was followed by two gel filtration steps resulting in a 98% pure IgM in about 30% yield. A somewhat modified procedure could also be used for purification of IgM from Cohn fraction II + III. The purified IgM was found to have a sedimentation constant in agreement with reported values. In immunoelectrophoresis and isoelectric focusing, purified IgM showed the same behaviour as IgM in plasma. Different fragments of IgM were tested for binding to the protamine-Sepharose adsorbent. IgM and Fab were not bound unlike (FC)5, indicating that the sites responsible for binding are located in the Fc part and that several Fc parts are necessary for sufficiently strong binding for adsorption.     

Purification of amylases and other enzymes by a forced-affinity chromatography method

An affinity matrix of soluble starch gel was prepared by cross-linking catalyzed by epichlorohydrin. The elution pattern of Taka-amylase A (TAA) indicated that the amount of enzyme bound to the starch gel column increased with increases in the ammonium sulfate (AmS) concentration in the equilibrating buffer. TAA had an affinity for the gels with a starch structure, and desorbed from the column with the buffer containing no AmS. Bound TAA was also eluted with starch and cyclodextrin solution. The AmS stimulative effect was partially replaced by polyethylene glycol and surfactants. Besides TAA, various, other amylases bound satisfactorily to the starch gel. Moreover, affinity purifications of dextranase, cellulase, and pectinase were done by gels with dextran, cellulose, and pectin structures, respectively. By the aid of forced effects of AmS, various carbohydrases could be purified by the affinity gels of polysaccharide linked by epichlorohydrin.     

Determination of ivermectin in human plasma by high-performance liquid chromatography

A specific reversed-phase HPLC-assay with sensitive fluorometric detection has been developed to measure the potent new antiparasitic agent ivermectin (CAS 70288-86-7) in human plasma (and urine). The lower limit of the method was 1 ng/ml and the intra-/interassay variability averaged 4.5/6.9%, respectively. The assay was applied for measuring plasma (urine) concentrations of ivermectin upto 56 (72 h) following a single oral dose of 6 and 12 mg. No unchanged or conjugated ivermectin could be detected in urine. Plasma concentrations increased linearly with dose but elimination half-life (12.6/13.4 h) was independent of the administered dose. Thus, the method is applicable for monitoring plasma levels during clinical and pharmacokinetic trials with ivermectin to evaluate its most efficacious dosage regimen.     

Assay of sulfonylureas in human plasma by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic procedure for the determination of chlorpropamide or tolbutamide in plasma in the presence of their metabolites is described. The ether extract of acidified plasma is redissolved in the mobile phase, 17% acetonitrile in 0.05 M aqueous ammonium formate, and chromatographed on a reverse-phase column on a high-performance liquid chromatograph fitted with a UV absorbance detector. Quantitation of plasma samples containing less than 0.5 mug/ml of chlorpropamide and 5 mug/ml of tolbutamide is reported, using these drugs as mutual internal standards. The retention times of the metabolites are such that they do not interfere in the procedure. The assay method was tested in a human volunteer with both drugs and found suitable for single-dose pharmacokinetic studies.     

Determination of dimethindene in human tears by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of dimethindene in human tears. The tear samples were diluted in a 0.01 M hydrochloric acid-n-propanol mixture to prevent the irreversible adsorption of dimethindene. The diluted samples were directly injected into the chromatographic system to avoid sample pretreatment. The validation data demonstrate that the method is specific, precise and accurate within the calibration range of 12 to 1000 ng/ml dimethindene free base.     

Purification of human factor IX by chromatography of a coagulation factor concentrate

A purification procedure for, and some properties of, coagulation factor IX are described. The coagulation factor concentrate used for the treatment of hemophilia B patients was employed as the starting material. The isolation procedure consists of chromatography in DEAE-cellulose, two chromatographies in hydroxyapatite gel and two gel filtrations in Sephadex G-200. Only trace amounts of factors II, VII and X were present in the final preparation and the specific activity of factor IX was 159 corresponding 10,300 times purification from plasma. The molecular weight was estimated to be 76,000 in gel filtration and 86,000 in sodium dodecyl sulfate disc gel electrophoresis. Three activity peaks with pIs 4.15, 4.25 and 4.40 were obtained by isoelectric focusing.     

Automated and sensitive method for the determination of formoterol in human plasma by high-performance liquid chromatography and electrochemical detection

An automated high-performance liquid chromatography (HPLC) method for the determination of formoterol in human plasma with improved sensitivity has been developed and validated. Formoterol and CGP 47086, the internal standard, were extracted from plasma (1 ml) using a cation-exchange solid-phase extraction (SPE) cartridge. The compounds were eluted with pH 6 buffer solution-methanol (70:30, v/v) and the eluate was further diluted with water. An aliquot of the extract solution was injected and analyzed by HPLC. The extraction, dilution, injection and chromatographic analysis were combined and automated using the automate (ASPEC) system. The chromatographic separations were achieved on a 5 microm, Hypersil ODS analytical column (200 mm x 3 mm I.D.), using (pH 6 phosphate buffer, 0.035 M + 20 mg/l EDTA)-MeOH-CH3CN (70:25:5, v/v/v) as the mobile phase at a flow-rate of 0.4 ml/min. The analytes were detected with electrochemical detection at an operating potential of +0.63 V. Intra-day accuracy and precision were assessed from the relative recoveries of calibration/quality control plasma samples in the concentration range of 7.14 to 238 pmol/l of formoterol base. The accuracy over the entire concentration range varied from 81 to 105%, and the precision (C.V.) ranged from 3 to 14%. Inter-day accuracy and precision were assessed in the concentration range of 11.9 to 238 pmol/l of formoterol base in plasma. The accuracy over the entire concentration range varied from 98 to 109%, and precision ranged from 8 to 19%. At the limit of quantitation (LOQ) of 11.9 pmol/l for inter-day measurements, the recovery value was 109% and C.V. was 19%. As shown from intra-day accuracy and precision results, favorable conditions (a newly used column, a newly washed detector cell and moderate residual cell current level) allowed us to reach a LOQ of 7.14 pmol/l of formoterol base (3 pg/ml of formoterol fumarate dihydrate). Improvement of the limit of detection by a factor of about 10 was reached as compared to the previously described methods. The method has been applied for quantifying formoterol in plasma after 120 microg drug inhalation to volunteers. Formoterol was still measurable at 24 h post-dosing in most subjects and a slow elimination of formoterol from plasma beyond 6-8 h after inhalation was demonstrated for the first time thanks to the sensitivity of the method.     

Determination of dopexamine hydrochloride in human blood by high-performance liquid chromatography with electrochemical detection

A method is described for the determination of dopexamine hydrochloride at concentrations of 5 to 100 ng/ml in human blood using electrochemical detection. The method uses a Hypersil ODS column and a mobile phase containing heptane sulphonate, orthophosphoric acid, diisopropylamine and disodium EDTA. Blood samples are stabilised immediately after collection by the use of dipotassium EDTA and a high concentration of sodium metabisulphite. The sample preparation procedure consists of a simple de-proteinisation with perchloric acid. The method is accurate, with inter-assay accuracies ranging from 100 to 104%, and is free of interference by blood from different individuals. Known and potential metabolites of dopexamine hydrochloride and a wide range of drugs do not interfere with the method. The method is precise with inter-assay coefficients of variation of 10.6% at 5 ng/ml and of less than 4.2% at higher concentrations. Stabilised blood samples may be stored for over six months at -25 degrees C prior to analysis.     

Determination of ofloxacin in human aqueous humour by high-performance liquid chromatography with fluorescence detection

This unusual pathology has not been described in the medical literature of the last ten years. A 39-year-old patient, affected by unilateral cryptorchidism, on the right side, and congenital inguinal hernia, reached the operating theatre suffering from occlusive intestinal syndrome, due to a clogged hernial sac. This clog was caused by a retracting testicle which in turn stopped the ileal ansa from slipping back in to the peritoneum. Through this case we can underline the excursus of such pathology, which isn't very frequent in the adult but can, nevertheless create a fairly serious pathology, often leading to neoplan.     

Determination of famotidine in human plasma by high performance liquid chromatography with column switching

While functional magnetic resonance imaging (fMRI) is now used widely for demonstrating neural activity-related signals associated with perceptual, motor, and cognitive processes in humans, to date this technique has not been developed for use with nonhuman primates. fMRI in monkeys offers a potentially valuable experimental approach for investigating brain function, which will complement and aid existing techniques such as electrophysiology and the behavioral analysis of the effects of brain lesions. There are, however, a number of significant technical challenges involved in using fMRI with monkeys. Here, we describe the procedures by which we have overcome these challenges to carry out successful fMRI experiments in an alert monkey, and we present the first evidence of activity-related fMRI signals from monkey cerebral cortex.     

Determination of pseudoephedrine hydrochloride and carbinoxamine maleate in combination drug formulation by liquid chromatography

The permeability to several chemical compounds and the histology of vaginal and buccal mucosa are very similar. Because vaginal mucosa is more abundant, it may be used as a model for the latter. To further develop the vaginal/buccal mucosa model, the objective of the present study was to evaluate the passage of a small polypeptide, vasopressin, across fresh and frozen specimens of these two mucosae. Specimens of fresh buccal and vaginal mucosa were taken from excised tissue obtained following vaginal hysterectomies and various oral surgical procedures. Pieces of buccal and vaginal tissue specimens obtained were used fresh or were snap-frozen and stored at -85 degrees C for periods of up to 10 months. Biopsies from fresh and thawed specimens were mounted in flow-through diffusion cells and their permeability to tritiated vasopressin was determined using a continuous flow-through perfusion system. Specimens were examined histologically before and after freezing as well as before and after permeability experiments and similarities between vaginal and buccal tissues verified. No statistically significant differences between flux values for fresh and frozen vaginal and buccal mucosa, respectively, were found. These results demonstrate that the permeation of vasopressin across fresh and frozen human vaginal and buccal mucosa is for practical purposes similar. These results further support the human vaginal/buccal mucosa model for in vitro permeability studies on therapeutically active compounds.     

Determination of human plasma xanthine oxidase activity by high-performance liquid chromatography

An assay for human plasma xanthine oxidase activity was developed with pterin as the substrate and the separation of product (isoxanthopterin) by high-performance liquid chromatography with a fluorescence detector. The reaction mixture consists of 60 microliters of plasma and 240 microliters of 0.2 M Tris-HCl buffer (pH 9.0) containing 113 microM pterin. With this assay, the activity of plasma xanthine oxidase could be easily determined despite its low activity. As a result, it could be demonstrated that the intravenous administration of heparin or the oral administration of ethanol did not increase plasma xanthine oxidase activity in normal subjects, and also that plasma xanthine oxidase activity was higher in patients with hepatitis C virus infection than in healthy subjects or patients with gout. In addition, a single patient with von Gierke's disease showed a marked increase in the plasma activity of this enzyme, relative to that apparent in normal subjects.     

Simultaneous determination of catechins in human saliva by high-performance liquid chromatography

Green tea extracts have been suggested to possess a preventive effect against dental caries. A quantitative method for their anticariogenic substances, catechins, was developed to evaluate their concentrations in human saliva after mouthrinsing with green tea extract. Salivary catechins were extracted to the organic phase after forming a complex with diphenylborate and an ion-pair with tetra-n-butylammonium, and then back-extracted to the acidic aqueous phase. The extract was analyzed by high-performance liquid chromatography using diode array detection at absorption wavelengths ranging from 269 to 278 nm. In reversed-phase chromatography by a gradient elution, eight catechins originating from green tea and an internal standard were separated in 15 min without interfering peaks. All the catechins were simultaneously and selectively determined in the concentration range 0.05-25.0 microg/ml. In replicate spiking experiments with standards, the mean recovery ranged between 86 and 99%, and both intra- and inter-assay C.V.s were within 2.3%. When mouthrinsing with an aqueous solution of green tea extract (5.0 mg/ml) containing eight catechins, the quantitative results revealed that each catechin was retained at microg/ml levels in saliva for up to 60 min.     

Purification of urokinase by affinity chromatography

Commercially available urokinase (EC 3.4.99.26), though highly active, is still contaminated with unrelated proteins and degradation fragments of urokinase. Further purification of a urokinase preparation by chromatography on benzamidine-Sepharose is described. The final preparation consisted of two molecular forms of urokinase with molecular weights of respectively 31 000 and 54 000. The 54 000-dalton urokinase appears to be composed of two protein chains, one of which is the 31 000-dalton urokinase. A monospecific antiserum against urokinase was raised.     

Assay of acyclovir in human skin layers by high-performance liquid chromatography

This paper describes an assay procedure for acyclovir quantification in human skin after in vitro transdermal transport experiments. The procedure employs warm distilled water for acyclovir (ACV) extraction and high-performance liquid chromatography (HPLC) as analytical method. The procedure has good reproducibility, sensitivity and specificity, resulting in a reliable method for biopharmaceutical studies of ACV distribution in skin tissue. The chromatographic conditions set up, using distilled water as mobile phase, makes the analytical procedure simple and easy to perform.